PROJECT SUMMARY/ABSTRACT Alcoholic liver disease (ALD) is one of the most common forms of chronic liver injury in the United States. Chronic ethanol consumption results in toxic metabolites in the liver and increased cell stress leading to inflammation and liver damage. Indeed, the cellular senescence occurred in hepatocytes, cholangiocytes and non-parenchymal cells - including hepatic stellate cells (HSCs) - is crucial to alcoholic metabolizing process. Despite the importance of steatosis in the disease process, the molecular pathways mediating ethanol metabolism, which is even more toxic than ethanol itself remain poorly understood. Small non-protein-coding RNAs (miRNAs) play a central role in various cellular pathways by regulating gene expression. Indeed, there is now compelling evidence for an association between ethanol metabolism and cellular senescence in ALDs. Although the combined evidence supports a link between miRNAs and alcoholic liver disease, there is a critical need to determine the underlying mechanism whereby ethanol-dependent miRNAs promote alcoholic liver injury. In the absence of such knowledge, the promise of developing novel mechanism-based preventive or treatment strategies for ALD will remain limited. Our long-term goal is to determine underlying mechanisms contributing to alcohol-induced liver disease so that new mechanism-based clinically effective prevention or treatment strategies can be developed. The objective for this proposal is to determine how ethanol-dependent miRNAs mediate cell metabolism and senescence in the progression of alcoholic liver diseases. Our central hypothesis is that ethanol-dependent miRNAs contribute to alcoholic liver injury through regulation of cellular metabolism and senescence in HSCs, hepatocytes and cholangiocytes. This hypothesis was formulated based upon the existing literature and our own preliminary data. The rationale for the proposed research is that a mechanistic understanding of the functional role of small non-coding RNAs mediated cell metabolism and senescence in alcoholic liver injury is likely to contribute to a conceptual framework whereby new targeted interventions to prevent or treat alcohol-induced liver disease. The following two specific aims are proposed: First, we will identify the downstream targets of miR-34a that involve in ethanol metabolism, cell survival and senescence. Second, we will determine the effects of microRNA mediated cellular senescence on alcoholic liver injury in senescence accelerated mice and miR-34a knockout mice in vivo. At the completion of the proposed research, it is our expectation to have determined fundamental mechanisms of miRNAs-regulated hepatic cell metabolism and different senescence properties in different hepatic cell lines (Aim 1). Further, we anticipate having quantified therapeutic effects of specific miRNAs during the recovery of alcoholic liver injury (Aim 2). These results are expected to have an important positive impact because a mechanistic understanding of the role ncRNAs play in alcohol-mediated liver disease is likely to provide a foundation for the development of evidence-based clinically useful approaches to treat or prevent ALD.